Abstract: Objective To investigate the maintenance of cell surface markers of keloid fibroblasts by hanging-drop culture method, and to preliminarily study the role of CD105 in regulating function of cells. Methods The fibroblasts were isolated from 3 keloid samples of ears, and the passage 5 cells were cultured using the adherent culture and the hanging-drop culture method for 1 week respectively. The passage 4 (P4) and 5 cells (P5) cultured by adherent culture method and the passage 5 cells cultured by hanging-drop culture method (P5HD)from the same patient were detected for the expression of cell surface markers including CD105, CD90, CD73 and CD44 by multicolor flow cytometry analysis. The mRNA expressions of surface markers and CTGF, Col IA1, Col IA2 were detected using real-time PCR. Results The positive cell percent of CD105+ and CD73+CD90+CD105+ in P4 and P5HD group were significantly higher than in P5 group by flow cytometry analysis, but there was no difference between P4 and P5HD group. The positive cell percent of CD73+, CD90+and CD44+ among three groups had no difference. The CD105 results of real-time PCR were in accordance with that of flow cytometry analysis. The mRNA expression tendency of CTGF and ColIA1 was similar to CD105. Conclusion The hanging-drop culture method is helpful to maintain the expression of CD105 surface marker and functional genes related to fibrosis and collagen production, then to keep the biological function of keloid fibroblasts, but the underlying mechanism needs to be discovered.

Key words: Keloid, Fibroblasts, Hanging-drop culture, Cluster of differentiation 105